The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells.

Bladder cancer (BC) is a malignant tumor of the urinary system with high mortality and recurrence rates. Proteasome subunit type 4 (PSMB4) is highly expressed and has been identified as having oncogenic properties in a variety of cancer types. This study aimed to explore the effect of PSMB4 knockdown on the survival, migration, and angiogenesis of human bladder cancer cells with different degrees of malignancy. We analyzed the effects of PSMB4 knockdown in bladder cancer cells and endothelial cells in the tumor microenvironment. PSMB4 was highly expressed in patients with low- and high-grade urothelial carcinoma. Inhibition of PSMB4 reduced protein expression of focal adhesion kinase (FAK) and myosin light chain (MLC), leading to reduced migration. Furthermore, the suppression of PSMB4 decreased the levels of vascular endothelial factor B (VEGF-B), resulting in lower angiogenic abilities in human bladder cancer cells. PSMB4 inhibition affected the migratory ability of HUVECs and reduced VEGFR2 expression, consequently downregulating angiogenesis. In the metastatic animal model, PSMB4 knockdown reduced the relative volumes of lung tumors. Our findings suggest the role of PSMB4 as a potential target for therapeutic strategies against human bladder cancer.

Hematopoietic stem cell transplantation in a patient with proteasome-associated autoinflammatory syndrome (PRAAS).

BACKGROUND: Proteasome-associated autoinflammatory syndromes (PRAASs) form a family of recently described rare autosomal recessive disorders of disturbed proteasome assembly and proteolytic activity caused by mutations in genes coding for proteasome subunits. The treatment options for these proteasome disorders consist of lifelong immunosuppressive drugs or Janus kinase inhibitors, which may have partial efficacy and noticeable side effects. Because proteasomes are ubiquitously expressed, it is unknown whether hematopoietic stem cell transplantation (HSCT) may be a sufficient treatment option. OBJECTIVE: Our aim was to report the case of a young boy with a treatment-resistant cutaneous vasculitis that was initially suspected to be associated with a gene variant in SH2D1A. METHODS: Whole-exome sequencing was performed to identify the genetic defect. Molecular and functional analyses were performed to assess the impact of variants on proteasomal function. The immune characterization led to the decision to perform HSCT on our patient and conduct follow-up over the 7-year period after the transplant. Because loss of myeloid chimerism after the first HSCT was associated with relapse of autoinflammation, a second HSCT was performed. RESULTS: After the successful second HSCT, the patient developed mild symptoms of lipodystrophy, which raised the suspicion of a PRAAS. Genetic analysis revealed 2 novel heterozygous variants in PSMB4 (encoding proteasomal subunit ß7). Retrospective analysis of patient cells stored before the first HSCT and patient cells obtained after the second HSCT demonstrated that HSCT successfully rescued proteasome function, restored protein homeostasis, and resolved the interferon-stimulated gene signature. Furthermore, successful HSCT alleviated the autoinflammatory manifestations in our patient. CONCLUSION: Patients with treatment-resistant PRAAS can be cured by HSCT.

Interference with PSMB4 Expression Exerts an Anti-Tumor Effect by Decreasing the Invasion and Proliferation of Human Glioblastoma Cells.

BACKGROUND/AIMS: Glioblastoma (GBM) is a malignant brain tumor with a poor prognosis. Proteasome subunit beta type-4 (PSMB4) is an essential subunit that contributes to the assembly of the 20S proteasome complex. However, the role of PSMB4 in glioblastomas remains to be clarified. The aim of this study was to investigate the role of PSMB4 in GBM tumor progression. METHODS: We first analyzed the PSMB4 protein and mRNA expression in 80 clinical brain specimens and 77 datasets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Next, we inhibited the PSMB4 expression by siRNA in cellular and animal models to explore PSMB4's underlying mechanisms. The cell survival after siPSMB4 transfection was assayed by MTT assay. Annexin V and propidium iodide staining was used to monitor the apoptosis by flow cytometric analysis. Moreover, the migration and invasion were evaluated by wound healing and Transwell assays. The expression of migration-related and invasion-related proteins after PSMB4 inhibition was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of PSMB4 knockdown in the in vivo study. RESULTS: Basis on the results of bioinformatics study, glioma patients with higher PSMB4 expression had a shorter survival time than those with lower PSMB4 expression. The staining of clinical brain tissues showed elevated PSMB4 expression in GBM tissues compared with normal brain tissues. The PSMB4 inhibition decreased proliferation, migration and invasion abilities in human GBM cells. Downregulated PSMB4 resulted in cell cycle arrest and apoptosis in vitro. In an orthotropic xenograft mouse model, the glioma tumors progression was reduced when PSMB4 was down-regulated. The decreased PSMB4 enhanced the anti-tumor effect of temozolomide (TMZ) on tumor growth. In addition, the absence of PSMB4 decreased the expression of phosphorylated focal adhesion kinase and matrix metallopeptidase 9 in vivo. CONCLUSION: PSMB4 inhibition in combination with TMZ may exert an anti-tumor effect by decreasing cell proliferation and invasion as well as by promoting apoptosis in human glioblastoma cells. This research may improve the therapeutic efficacy of glioblastoma treatment.

Upregulation of PSMB4 is Associated with the Necroptosis after Spinal Cord Injury.

Spinal cord injury (SCI) is one of the most common and severe complications in spine injury. It is difficult to prevent cell necroptosis and promote the survival of residual neurons after SCI. Proteasome beta-4 subunit (PSMB4) is the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo, and our previous study showed that PSMB4 is significantly associated with neuronal apoptosis in neuroinflammation. However, PSMB4 function in the necroptosis after SCI is unkown. RIP3, a key regulatory factor of necroptosis, correlates with the induction of necroptosis in various types of cells and signaling pathway. Upregulation of the RIP3 expression may play a role as a novel molecular mechanism in secondary neural tissue damage following SCI. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of PSMB4 during the pathological process of SCI. We found PSMB4 expression was significantly up-regulated 3 days after injury by western blot and immunohistochemical staining. Double immunofluorescent staining indicated obvious changes of PSMB4 expression occurred in neurons. Significant up-regulation of PSMB4 expression was observed in Rip3 positive neurons at 3 days after SCI, which indicated that PSMB4 might play a vital role in the regulation of Rip3. Overexpress and knockdown PSMB4 could intervene the RIP3 and Mixed lineage kinase domain-like protein (MLKL) pathway in Tumor necrosis factor-α (TNF-α) induced necroptosis cell model. Based on our experimental data, we boldly conclude that PSMB4 is associated with RIP3 involved necroptosis after SCI.

PSMB4 expression associates with epithelial ovarian cancer growth and poor prognosis.

PURPOSE: In this study, we investigated the expression and role of PSMB4 in human epithelial ovarian cancer(EOC). METHODS: Western blot was used to evaluate the expression of PSMB4 in EOC tissues, and immunohistochemical analysis was performed on 115 cases of ovarian cancers. Then, we used Fisher exact test to analyze the correlation between PSMB4 and clinicopathological parameters. Starvation and re-feeding assay was used to assess cell cycle. CCK-8 assay and plate colony formation assay showed the influence of PSMB4 on proliferation of EOC cells. RESULTS: The expression of PSMB4 in EOC tissues was higher than normal ovary tissues and was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of PSMB4 was related to poor prognosis of EOC patients. Starvation and re-feeding assay suggested that PSMB4 played a critical role in EOC cell proliferation. CCK-8 assay and plate colony formation assay showed that EOC cells treated with PSMB4-siRNA reduced cell proliferation of EOC cells. Additionally, PSMB4 knockdown decreased NF-κB activity. PSMB4 also regulated the expression of NF-κB mediated proteins, including cyclin D1, and cyclin E which involved in cell proliferation. CONCLUSIONS: Our findings implied that PSMB4 is involved in the progression of EOC and could serve as potential therapeutical target of EOC. These data suggested that PSMB4 may promote cell proliferation via the NF-κB-target gene in EOC.

PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling.

Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expression of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies.

Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.

Proteasome dysfunction disrupts adipogenesis and induces inflammation via ATF3.

OBJECTIVE: Regulation of proteasomal activity is an essential component of cellular proteostasis and function. This is evident in patients with mutations in proteasome subunits and associated regulators, who suffer from proteasome-associated autoinflammatory syndromes (PRAAS). These patients display lipodystrophy and fevers, which may be partly related to adipocyte malfunction and abnormal thermogenesis in adipose tissue. However, the cell-intrinsic pathways that could underlie these symptoms are unclear. Here, we investigate the impact of two proteasome subunits implicated in PRAAS, Psmb4 and Psmb8, on differentiation, function and proteostasis of brown adipocytes. METHODS: In immortalized mouse brown pre-adipocytes, levels of Psmb4, Psmb8, and downstream effectors genes were downregulated through reverse transfection with siRNA. Adipocytes were differentiated and analyzed with various assays of adipogenesis, lipogenesis, lipolysis, inflammation, and respiration. RESULTS: Loss of Psmb4, but not Psmb8, disrupted proteostasis and adipogenesis. Proteasome function was reduced upon Psmb4 loss, but partly recovered by the activation of Nuclear factor, erythroid-2, like-1 (Nfe2l1). In addition, cells displayed higher levels of surrogate inflammation and stress markers, including Activating transcription factor-3 (Atf3). Simultaneous silencing of Psmb4 and Atf3 lowered inflammation and restored adipogenesis. CONCLUSIONS: Our study shows that Psmb4 is required for adipocyte development and function in cultured adipocytes. These results imply that in humans with PSMB4 mutations, PRAAS-associated lipodystrophy is partly caused by disturbed adipogenesis. While we uncover a role for Nfe2l1 in the maintenance of proteostasis under these conditions, Atf3 is a key effector of inflammation and blocking adipogenesis. In conclusion, our work highlights how proteasome dysfunction is sensed and mitigated by the integrated stress response in adipocytes with potential relevance for PRAAS patients and beyond.

PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion (I/R) injury induces cardiomyocyte apoptosis to deteriorate heart function. Thus, how to inhibit cardiomyocyte apoptosis is the focus of recent researches. Proteasome family member PSMB4 (proteasome subunit beta type-4) promotes cell survival. The relationship between PSMB4 and cardiomyocyte apoptosis during myocardial I/R is unknown. In this study, PSMB4 expression increased in rat myocardial I/R model, positively correlated with cleaved caspase-3 expression, negatively correlated with Bcl-2 expression. In vitro, neonatal ventricle cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed to mimic myocardial I/R. PSMB4 silence promoted cardiomyocyte apoptosis and IκBα expression, inhibited the activation of NF-κB. On the contrary, PSMB4 overexpession inhibited cardiomyocyte apoptosis and IκBα expression, promoted the activation of NF-κB. Additionally, PSMB4-IκBα interaction was identified, suggesting that PSMB4 might participate in the proteasome dependent degradation of IκBα. The data indicates that PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial I/R, which can supply novel molecular target for the treatment of ischemic heart disease.

Up-regulation of PSMB4 is associated with neuronal apoptosis after neuroinflammation induced by lipopolysaccharide.

Proteasomes are major intracellular extralysosomal organelle for protein degradation and a central source of antigenic peptides in the endogenous pathway. Proteasome beta-4 subunit (PSMB4) was recently identified as potential cancer driver genes in several tumors. However, information regarding its regulation and possible function in the central nervous system is still limited. The present study was designed to elucidate dynamic changes in PSMB4 expression and distribution in the cerebral cortex in a lipopolysaccharide (LPS)-induced neuroinflammation rat model. It was found that PSMB4 expression was increased significantly in apoptotic neurons in the brain cortex after LPS injection. Moreover, there was a concomitant up-regulation of active caspase-3, cyclin D1, and CDK4 in vivo and vitro studies. In addition, these three proteins in cortical primary neurons were decreased after knocking down PSMB4 by siRNA. Collectively, these results suggested that PSMB4 may be involved in neuronal apoptosis in neuroinflammation after LPS injection.

Predictive value of PAK6 and PSMB4 expression in patients with localized prostate cancer treated with dose-escalation radiation therapy and androgen deprivation therapy.

PURPOSE: The present study analyzed the expression by immunochemistry of the novel markers P21-activated protein kinase 6 (PAK6) and proteasome beta-4 subunit (PSMB4) in men with localized prostate cancer (PC) who were treated with dose-escalation radiotherapy (RT) and androgen deprivation therapy. MATERIALS AND METHODS: Between 1996 and 2004, a cohort of 129 patients with PC who underwent diagnostic biopsies pretreatment and 24 to 36 months following RT were enrolled in this study. Suitable archival diagnostic tissue was obtained from 89 patients. Median follow-up was 129 months (48-198). Correlation analysis was done to assess association between PAK6 and PSMB4 expression and clinical outcome. RESULTS: PAK6 and PSMB4 were expressed in the cytoplasm in 62% and 96.7% of diagnostic biopsies, respectively. Increased staining for PAK6 was significantly (P = 0.04) correlated with higher Gleason scores. In the multivariate analysis, the intensity of PSMB4 staining was an independent predictor of local relapse (hazard ratio = 8.6, P = 0.04). CONCLUSIONS: To our knowledge, this is the first description of PAK6 and PSMB4 expression in the diagnostic specimens of men with PC who were treated with RT. If confirmed by further studies, increased expression of these genes could be used to identify patients at a high risk of developing local failure following high-dose RT, thus better tailoring treatments for the individual patient.